ApoA-I is the major protein component of HDL and is required both for the biogenesis and the functions of HDL. Lipid-free apoA-1 and different HDL species formed by sequential lipidation of apoA-I by ABCA1 appear to have distinct functions in cholesterol efflux, selective uptake of lipids, activation of LCAT, and possibly other functions of HDL. It is our hypothesis based on our recent findings, that subtle changes in the apoA-l structure may affect HDL biogenesis and functions including ABCA1- and SR-BI-mediated cholesterol efflux, SR-Bl-mediated selective uptake of lipids, and activation of the LCAT. In this application we will use in vitro and in vivo approaches to elucidate the structure and functions of apoA-I. The in vitro studies will utilize mutant forms of apoA-I and HDL produced in apoA-l-/- mice following adenovirus infection. The in vivo studies will utilize adenovirus-mediated gene transfer in apoA-l-/- mice and transgenic mice expressing apoA-l mutants. Our specific aims are: 1) To determine by physicochemical methods the contribution of specific domains and residues of apoA-l that are responsible for stabilizing the conformation and structure of apoA-l through intra- or intermolecular interactions in solution, or when bound to lipids. Structural changes of the apoA-I mutants will be correlated with the in vitro and in vivo functions of apoA-l, including LCAT activation and lipid and lipoprotein binding. 2) To investigate the functional interactions of lipid-bound apoA-l with SR-Bl and the effect of apoA-l mutations in SR-Bl-mediated cholesterol efflux and selective lipid uptake. 3) To investigate the effect of apoA-I mutations in cholesterol efflux and the functional interactions of lipid-free apoA-I with ABCA1 that leads to efflux of cellular phospholipid and cholesterol. 4) To investigate the implications of apoA-l mutations on the biogenesis and the function of different HDL species using adenovirus-mediated gene transfer in apoA-l-/- mice as well as transgenic mice. Epidemiological and genetic data, combined with recent transgenic experiments, suggest that increased apoA-I and HDL levels protect from atherosclerosis. In contrast, low apoA-I and HDL levels predispose humans to coronary artery disease (CAD), a leading cause of mortality worldwide. Understanding the molecular structure and the various biological functions of apoA-I may lead to new pharmacological approaches to prevent and/or treat these conditions.